The small molecule cyclic di-GMP (c-di-GMP) is known to affect bacterial gene expression in myriad ways. In Vibrio cholerae in vivo, the presence of c-di-GMP together with the response regulator VpsR results in transcription from PvpsL, a promoter of biofilm biosynthesis genes. VpsR shares homology with enhancer binding proteins that activate sigma54-RNA polymerase, but it lacks conserved residues needed to bind to sigma54-RNA polymerase and to hydrolyze ATP, and PvpsL transcription does not require sigma54 in vivo. Consequently, the mechanism of this activation has not been clear. Using an in vitro transcription system, we have demonstrated activation of PvspL in the presence of VpsR, c-di-GMP, and sigma70-RNA polymerase. c-di-GMP does not significantly change the affinity of VpsR for PvpsL DNA or the DNase I footprint of VpsR on the DNA, and it is not required for VpsR to dimerize. However, DNase I and KMnO4 footprints reveal that the sigma70-RNA polymerase / VpsR/c-di-GMP complex on PvpsL adopts a different conformation from that formed by sigma70-RNA polymerase alone, with c-di-GMP, or with VpsR. Our results suggest that c-di-GMP is required for VpsR to generate the specific protein-DNA architecture needed for activated transcription, a previously unrecognized role for c-di-GMP in gene expression.